Docosahexaenoic acid (DHA) impairs hypoxia-induced cellular and exosomal overexpression of immune-checkpoints and immunomodulatory molecules in different subtypes of breast cancer cells

Background: Tumor cells express immune-checkpoint molecules to suppress anti-tumor immune responses. In part, immune evasion takes place by secreting exosomes bearing immune-checkpoint and immunomodulatory molecules and their inducing and/or regulating agents e.g., microRNAs (miRs). This study aimed to evaluate the effects of omega-3 fatty acid, docosahexaenoic acid (DHA), on the expression of some selected immune-checkpoint and immunomodulatory molecules and their regulating miRs under both normoxic and hypoxic conditions in triple negative (TNBC) invasive and triple positive non-invasive breast cancer cell lines. Methods: MDA-MB-231 and BT-474 cells were treated with 100 µM DHA under hypoxic and normoxic conditions for 24 h. Exosomes were isolated by ultracentrifuge and confirmed by electron microscope and anti-CD9, -CD63, -CD81 immunoblotting. Total RNA from cells and exosomes were extracted and expression of CD39, CD73, CD47, CD80, PD-L1, B7-H3, B7-H4 genes and their related miRs were evaluated by quantitative Real-time PCR. Results: This study showed significant over-expression of immune-checkpoint and immunomodulatory molecules under hypoxic condition. Treatment with DHA resulted in a significant decrease in immune-checkpoint and immunomodulatory molecule expression as well as an upregulation of their regulatory miRNA expression. Conclusion: DHA supplementation may be utilized in breast cancer therapy for down-regulation of cellular and exosomal immune escape-related molecules.